In the recent past, light microscopic methods have been developed with which, based on a sequential, stochastic localization of individual point objects, in particular fluorescence molecules, image structures can be imaged that are smaller than the diffraction-dependent resolution limit of conventional light microscopes. Such methods are, for example, described in WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1, US 2009/0134342 A1; DE 10 2008 024 568 A1; “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)”, Nature Methods 3, 793-796 (2006), M. J. Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching”, Geisler C. et al, Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as localization microscopy. The applied methods are known in the literature, for example, under the designations (F)PALM ((Fluorescence) Photoactivation Localization Microscopy), PALMIRA (PALM with Independently Running Acquisition), GSD(IM) (Ground State Depletion (Individual Molecule return) Microscopy) or (F)STORM ((Fluorescence) Stochastic Optical Reconstruction Microscopy).
The new methods have in common that the structures to be imaged are prepared with markers that have two distinguishable states, namely a “bright” state and a “dark” state. When, for example, fluorescent dyes are used as markers, then the bright state is a state in which they are able to fluoresce and the dark state is a state in which they are not able to fluoresce. For imaging image structures with a resolution that is higher than the conventional resolution limit of the imaging optical system, a small subset of the markers is repeatedly brought into the bright state and thus it is so to speak activated. In this connection, the activated subset is to be chosen such that the average distance of adjacent markers in the bright state is greater than the resolution limit of the imaging optical system. The luminance signals of the activated subset are imaged onto a spatially resolving light detector, e.g. a CCD camera. Thus, of each marker a light spot is detected whose size is determined by the resolution limit of the imaging optical system.
In this way, a plurality of raw data single frames is captured, in each of which a different activated subset is imaged. Using an image analysis process, then in each raw data single frame the centroids of the light spots are determined which represent those markers that are in the bright state. Thereafter, the centroids of the light spots determined from the raw data single frames are combined to a total representation. The high-resolution image created from this total representation reflects the distribution of the markers. For a representative reproduction of the structure to be imaged sufficient signals have to be detected. Since however the number of markers in the respective activated subset is limited by the minimum average distance which two markers may have in the bright state, a great many raw data single frames have to be captured to completely image the structure. Typically, the number of raw data single frames is in a range between 10,000 and 100,000.
The time required for capturing one raw data single frame has a lower limit that is predetermined by the maximum image capturing rate of the imaging detector. This results in relatively long total capturing times for a series of raw data single frames required for the total representation. Thus, the total capturing time can take up to several hours.
Over this long total capturing time, a movement of the specimen to be imaged relative to the imaging optical system may occur. Since for creating a high-resolution total image all raw data single frames are combined after the determination of the centroids, each relative movement between specimen and imaging optical system that occurs during the capturing of two successive raw data singles frames impairs the spatial resolution of the total image. In many cases, this relative movement results from a systematic mechanical movement of the system, also referred to as mechanical drift which is caused, for example, by thermal expansion or shrinkage, by mechanical strains or by the change in the consistency of lubricants used in the mechanical components.
In the above-described high-resolution methods it is of particular importance to provide for a drift-free positioning of the specimen on a microscope stage. In the prior art, often so-called mechanical stages (X-Y stages) are used for this purposes, which allow to move a specimen holder on a platform in two orthogonal directions (in the following also referred to as X and Y direction) in a plane of displacement (X-Y-plane) that is parallel to the platform. Such a mechanical stage consists of two superposed plates which are mechanically coupled to each other as well as a drive for moving the two plates against each other. Thus, in the case of a mechanical stage the movements of the specimen holder in the direction of the X-axis and the Y-axis are coupled to each other.
By means of such a microscope stage it is basically possible to position the specimen holder on the microscope stage in an easy and precise manner. However, due to the mechanical coupling of the components forming the mechanical stage a mechanical drift occurring in one of these components as a result of thermal influences or mechanical strains existing in the drive also has an effect on the respective other components. In high-resolution light microscopic methods in which long total capturing times are intended and resolutions in the nanometer range are aimed at, this may result in intolerable image shifts.
From DE 695 30 095 T2, a planar positioning stage is known having a platform upon which a workpiece to be positioned is placed. The positioning stage further comprises a first and a second actuator means which are adapted to move the workpiece in an actuating plane along a first axis or along a second axis that runs transversely to the first axis.
In DE 35 14 431 A1 a microscope stage drive is described which comprises two carriages which are movable orthogonally to each other, and coaxially mounted drive members for moving the carriages. The force transmission between the drive members and the carriages takes place via traction means running over deflection rollers.
From DE 19 38 771 A, a precision drive for two carriages is known which can be guided and displaced orthogonally to each other, of which a first carriage is mounted in a stationary guide and a second carriage is mounted in a guide provided on the first carriage.
With respect to the prior art, reference is further made to JP 2005 091 866 A, U.S. Pat. No. 5,000,554 A, JP 58-106 514 A and US 2006/0 138 871 A1, from which positioning devices are known with which an object can be moved in a plane of displacement along two axes which are orthogonal to each other.